The major objective of this project is to develop a series of flagellar motility mutants of Chlamydomonas reinhardtii defective in the assembly and/or enzymatic activities of axonemal dynein-ATPase containing doublet microtuble arms. Over 100 nonconditional and thermosensitive flagellar motility mutants have already been isolated from which to screen for potentially interesting mutants. We anticipate obtaining additional mutants with frameshift mutagens. Mutants likely to meet the objectives of this project will be initially identified on the basis of quantitative deficiencies in Mg ions-activated ATPase activity in isolated axonemal preparations. ATPase-defective mutants will be characterized at the genetic level to determine their segregation patterns, allelism by recombination analysis, phenotypes dominance and complementation in temporary dikaryons, and map location based on linkage to established markers. Electron microscopic studies will be undertaken to identify mutations leading to assembly defects in the dynein arms. At the same time, chemical analysis of (35S) sulfate-labeled axonemal components resolved by 2-dimensional and 1-dimensional gel electrophoresis will be pursued to identify missing or altered polypeptides in the mutants. Available procedures for fractionation of the polypeptides and dynein-ATPase activities which are solubilized with extraction of the dynein arms will be used to resolve the specific structural and/or enzymatic alterations in the mutants. Two complementary experimental approaches will be used to identify the mutant gene products - molecular rescue in temporary quadriflagellate dikaryons and chemical analysis of induced revertants. If appropriate mutants are identified, structural analysis of wild-type and variant gene products will be pursued. The ultimate objective of this project is to utilize a genetic approach to define the molecular requirements for the assembly and function of the dynein arms in generating active sliding displacements of microtubules.